Screening X. tropicalis using Gynogenesis
Gynogenesis is a genetic technique that manipulates the ploidy of embryos. Breaking the word into its roots, “gyno” meaning of or relating to the female and “genesis” meaning creation, gynogensis is the creation of embryos using the female's DNA only. Paternal contribution is nullified by irradiating the sperm with ultraviolet light, wherein the DNA is not transmittable, but the cellular signals exist to activate fertilization. By performing the technique at the appropriate critical time, the ploidy of haploid embryos is increased to make the embryo diploid. This results in two copies of any recessive alleles, thus unmasking any mutation instantly, and removing the need for multiple, tedious crosses and generation times that have been used historically to reveal recessive traits.
There are two types of gynogenesis: early gynogenesis and late gynogenesis. Early gynogenesis prevents the polar body extrusion during the second metaphase of meiosis, making the loci near the centromere homozygous, while the distal-most portions of the chromosome are still subject to crossing-over and may not become completely homozygous. Late gynogenesis prevents the first mitotic division, thereby making all loci along the chromosome homozygous because crossing-over has already occurred. Either method will unmask the recessive traits, but X. tropicalis embryos tolerate early gynogenesis much better than the late gynogenesis.
Two techniques now exist to perform gynogenesis. The historical method, developed by Tompkins and Reinschmidt , uses hydrostatic pressure to suppress polar body extrusion or mitotic cleavage. However, the use of hydrostatic pressure requires bulky, expensive equipment and there are many artifactual defects and high death rates observed. The method developed in our laboratory uses a cold shock technique to achieve the same ends, while eliminating the need for expensive equipment, improving the viability of the embryos, and decreasing the number of background defects observed. We have also had success with producing “late cold shock” diploids that have survived to adulthood.
The cold shock method has been extensively tested and validated. After the parameters were determined, an experiment using a gamma-crystallin GFP male was used to demonstrate that paternal contribution had been nullified. ( GFP experiment ) Once the paternal absence was verified, the method was used to reveal the phenotype of one of our established mutants, further validating the method. A simultaneous comparison of the hydrostatic pressure method (often referred to as the “bomb”) and the cold shock method on the eggs of the same female revealed that the cold shock method is at least twice as efficient as the “bomb” method, on average, in producing viable, normal embryos.
Early cold shock gynogenesis protocol
Late cold shock gynogenesis protocol
Hydrostatic pressure gynogenesis protocol